Interferons (IFNs) were discovered in 1957 by Isaacs and Lindenmann (Proc R Soc Lond B Biol Sci 147(927):258-67) and were named for their ability to interfere with viral proliferation. Interferons can also combat bacterial and parasitic infections and promote a wide range of activities, including inhibition of cell growth, differentiation, and spontaneous apoptosis, and modulation of the immune system, including promotion of antigen presentation, induction of cytokine production, and promotion of T cell maturation and clonal propagation. IFNs were originally classified by their sources: leukocytes IFN-α-1 (SEQ ID NO: 1), and IFN-α-2 (SEQ ID NO: 2), fibroblasts IFN-β (SEQ ID NO: 3), and immune cells IFN-γ (SEQ ID NO: 4).
Type I and Type II Interferons are defined based on their receptor specificity. Type I interferons are a family of monomeric proteins including IFNα, IFNω, IFNβ, and IFNτ that has been described only in ungulate species. The only known type II interferon is the dimeric IFNγ.
Human type I interferons number 13 distinct non-allelic alpha proteins, one beta, and one omega. Each member is a mature protein of 165 or 166 amino acid residues, all sharing the same structure, having 2 conserved disulfide bonds, Cys1-Cys98 and Cys29-Cys138, and binding the same cell surface receptor, IFNAR.
A high level of sequence homology (80%) is displayed among the various interferon alpha sub-types, and about 35% homology exists between these sub-types and IFNβ. In spite of the high homology of the different sub-types, their biological activities differ notably. For example, IFNβ has a much higher anti-proliferative activity then IFNα proteins, due to the tighter binding of IFNβ to the IFNAR1 receptor subunit.
All type I interferons signal through a common receptor complex known as “interferon receptor 1” (“IFNAR”), in the context of a “ternary complex” containing IFN and the subunits IFNAR1 and IFNAR2. Binding of IFNα2 to IFNAR1 alone is relatively weak, namely, 1.5-5 μM, in contrast to the ˜10 nM binding affinity for IFNAR2. The formation of the ternary complex occurs in a sequential mode, with an intermediate complex first forming between interferon and IFNAR2, followed by the recruitment of IFNAR1 in a 1:1:1 stoichiometry. Because of cooperative binding to the 2 surface-bound receptor subunits, the apparent ligand binding affinity is higher than binding to IFNAR2 alone, and depends on the relative and absolute receptor surface concentrations (Lamken, P. et al., 2004, J Mol Biol 341, 303-318). Association of IFNAR1 and IFNAR2 stimulates the activation of the constitutively-associated intracellular kinases Jak1 and Tyk2, leading to a tyrosine phosphorylation cascade that results in the dimerization and transport into the nucleus of the signal transducers and activators of transcription (STATs), where they bind to specific DNA sequences and stimulate transcription of hundreds of responsive genes.
The structures of several type I interferons are known, including murine IFNβ (1IFA, 1RMI), human IFNβ (1AU1), human IFNα2 (1RH2, 1ITF), and ovine IFNτ (1B5L). Structurally, interferons are members of the α-helical cytokine family. The extracellular part of IFNAR2 consists of 2 immunoglobulin-like domains, with the IFNα2 binding site located at the N-terminal domain and the connecting loop. Mature IFNAR1 is a 530 amino acid protein, with a 21-residue transmembrane segment and a 100-residue cytoplasmic domain. The sequence suggests that its extracellular portion is composed of 4 immunoglobulin-like domains. The IFNAR1 binding site of IFNβ is located on the B, C, and D helices and the DE loop, in the case of IFNα2, it is located on helices B and C (Hu R et al, Human IFN-alpha protein engineering: the amino acid residues at positions 86 and 90 are important for antiproliferative activity. J Immunol 167(3):1482-9, 2001).
In general, IFNβ exhibits an additional repertoire of activities over IFNα. IFNβ has generally higher activity in transcriptionally activating IFN responsive genes, and is active at lower concentrations than IFNα. Affinity for IFNAR1 is likely to be the key difference between IFNα2 and IFNβ (Jaitin, 2006. Mol Cell Biol. 26, 1888-1897).
Recombinant IFNα2 protein exhibits anti-cancer effects. However, IFNα2 treatment is not always effective and can result in intolerable side effects. WO 97/12630 and WO 01/54678 disclose treatment of cancer patients with a temozolomide/IFN combination.
Hepatitis C virus (HCV) is the most common chronic blood-borne infection in the United States. Chronic liver disease is the tenth leading cause of death among adults in the United States, and an estimated 40% of chronic liver disease is HCV-related. HCV-associated end-stage liver disease is the most frequent indication for liver transplantation among adults. Antiviral therapy of chronic hepatitis C with recombinant IFN has improved rapidly over the last decade. Nevertheless, even with the latest regimens, combination therapy with pegylated IFN-α plus ribavirin, 40% to 50% of patients fail therapy, i.e. do not respond or relapse. These patients have no effective therapeutic alternative.
There remains an unmet medical need for IFNAR agonists of enhanced potency for treating cancer and infectious disease, e.g. HCV.
Type I diabetes, also known as autoimmune diabetes or insulin-dependent diabetes mellitus (IDDM), is an autoimmune disease characterized by the selective destruction of pancreatic cells by autoreactive T lymphocytes. IFN-α has also been implicated in the pathogenesis of systemic lupus erythematosus (SLE) (Ytterberg and Schnitzer, Arthritis Rheum 25: 401-406, 1982). IFN-α has been implicated in IDDM pathogenesis; thus, antagonists of IFN-α and -β signaling are useful in treating IDDM and SLE, as disclosed in WO 93/04699 and WO 02/066649, respectively.
There remains an unmet medical need for potent IFNAR antagonists for treating autoimmune diseases, e.g. IDDM.
Multiple sclerosis (MS) is a chronic, neurological, autoimmune disease that can cause impaired vision and muscular degeneration. IFN-β-1a and β-1b are both approved for treating MS. Therapeutic effect of IFN-β is likely to be due to skewing of the T-cell response towards a type 2 phenotype (Zafranskaya M et al, Interferon-beta therapy reduces CD4+ and CD8+ T-cell reactivity in multiple sclerosis. Immunology 121(1):29-39, 2007).
LE is an autoimmune disease that causes inflammation and damage to various body tissues and parts, including joints, kidneys, heart, lungs, brain, blood vessels, and skin. The most common symptoms of LE include achy or swollen joints (arthritis), fever, prolonged or extreme fatigue, skin rashes and kidney problems. The goals of effective treatment of LE are to prevent flares, minimize organ damage and complications, and maintain normal bodily functions. Because of the limited success of currently available medications and their potentially serious side effects, it is important to provide an alternative effective treatment for LE.
There remains an unmet medical need for IFN-β analogues of enhanced potency for treating MS and other auto-immune diseases with similar pathology.
US Patent Application Publication No. 2004/0230040 discloses IFN-α2 variants with added cysteine residues. US Patent Application Publication No. 2004/0002474 discloses α interferon homologues with anti-proliferative activity. These IFN-α2 variants are unrelated to the IFN-α2 variants of the present invention.